WO1990009453A1

WO1990009453A1 - Process and device for rapid detection of microorganisms in samples

Info

Publication number: WO1990009453A1
Application number: PCT/DE1990/000081
Authority: WO
Inventors: Dieter Naumann; Harald Labischinski
Original assignee: Bruker Analytische Messtechnik Gmbh
Priority date: 1989-02-09
Filing date: 1990-02-08
Publication date: 1990-08-23
Also published as: DE59000762D1; EP0456695B1; US5660998A; DE3903777A1; EP0456695A1

Abstract

The invention concerns a process and a device for rapid detection and enumeration of microorganisms. The test sample is briefly incubated, and the microcolonies whose growth on a nutrient medium plate cannot be yet detected with the naked eye are transferred directly to a sample carrier by means of special impression techniques and detected spectroscopically. The detection itself is carried out by recording the spectra of all or a representative number of the impressions by means of an IR microscope. Not only can data concerning the number of microorganisms in the sample be obtained, but the detailed information contained in the spectra can be used for precise microbiological differentiation including species identification.

Description

description

Method for the rapid detection of microorganisms in samples and device for carrying out the method

The invention relates to a method for the rapid detection of microorganisms in a large number of samples, e.g. Urine, blood, water, food, pharmaceutical raw materials and products such as Ointments, liquid preparations, etc., which are generally free of such microorganisms, either n or where certain, fixed limit values for the total number of bacteria or also for individual, defined representatives of microorganisms should not be exceeded, and also a device to carry out the procedure »

Such detection work is carried out in the clinical area (sepsis, urinary tract infections, Li quortestung) and out of clinic area (assessment of the sterility or burden of starting materials and end products, such as water, milk, food etc.) to a large extent, because the results of such tests are of great clinical importance but are also of great importance for quality control, durability assessment and other product and production medium tests, for example in industrial companies. This is because of the frequent possibility of exponential growth of the microorganisms to be detected and the very early detection of, for example, infections to initiate therapeutic measures, which is often necessary in the clinical field, for example in the case of life-threatening sepsis or in the non-clinical area, for example in the case of Production of pharmaceuticals to prevent the uselessness of entire batches, for example when using contaminated starting materials with possibly considerable economic consequences, detection also possible in the same way ((currently ... B .. << T10 per ml or g sample) in possible required a short time.

When carrying out detection tests, the current state of the art generally uses either methods which allow very small and very specific very small amounts of certain individual groups of microorganisms to be detected, and methods which are less rapid and which are comparatively significantly larger Quantities of microorganisms (about> 10 bacteria per ml test sample) can detect a large number of different microorganisms in a non-specific manner (for an overview see, for example, in: Bergan, T .: Methods in Mi crobiol og, Vols. 14- 16, London, Orlando, San Diego, San Francisco, New York, Toron¬ to, Montreal, Sydney, Tokyo, Sao Paulo: Academic Press (1984); Kipps, TJ, Herzenberg, LA, in: Habermehl, K.-O .: Rapid Methods and Automation in Microbiology and Im unology, Berlin, New York, Tokyo: Springer Verlag (1985); Toorova, TP, Antonov, AS, in: Colwell, RR and Grigorova, R .: Methods in Microbiology, Vol. 19, London, Orlando, San Di ego, New York, Austin, Boston, Sydney, Tokyo, Toronto: Academic Press (1987); Thronsberry, C, in: Habermehl, K.-O .: Rapid Methods and Automation in Microbiology and Im- Munology, Berlin, New York, Tokyo: Springer Verlag (1985); Carlberg, DM, in: Lorian, V .: Antibiotics in Laboratory Medicine, Baltimore, London, Los Angeles, Sydney: Williams

Wilkiπs (1986)). The first group includes, in particular, molecular biological detection methods with which the detection indirectly by detecting a reaction of the sample, e.g. with monoclonal antibodies or also by DNA / DNA hybridization etc. Techniques. Characteristic of these techniques is the need for (expensive) generation and preparation of highly specific reagents for each individual type of the microorganism to be detected. It is advantageous in these methods, however, that a detection of the microorganisms already includes, depending on the specificity of the reagents used, a more or less accurate identification of the detected microorganism type, provided that cross-reactions with other organism types are a common problem , be ignored. Their use, which up to now has hardly been routine, is therefore foreseeably restricted to individual, particularly important (e.g. life-threatening germs in the event of an infection) organism groups. Representatives of the second group of processes typically include, e.g. microcalorimetric, conductivity / impedance and optical measurements.

With these methods, the sample to be examined for the presence of microorganisms, for example bacteria, is generally placed in a suitable culture medium which promotes the growth of the organisms, and this is incubated under the best possible growth conditions. The detection is then carried out, for example, by detecting the heat production of the culture by means of a microcalorimeter or the changes in the impedance of the culture fluid caused by the growth of the culture by means of suitable conductivity measuring systems. In a broader sense, this group of processes also includes simple optical measurements of the turbidity of the culture medium or the optical detection of microorganisms. The advantage of this group of processes lies in the relatively universal application to a large number of organism groups and, with the exception of the last-mentioned process variant, in the comparatively simple operability and automatability. On the other hand, the disadvantage is the lack of the possibility of parallel identification of the microorganisms, which, if necessary, has to be carried out separately using other methods, the relatively long time period between sample application and detection, which is due to cultivation, among other things, and the comparatively high detection limits (e.g. approx. 10 Cells / ml urine in the case of urological specimens which are particularly common in medical practice).

DE-Al-32 47 891 describes a method for identifying microorganisms based on the image analysis evaluation of infrared spectra in selected, small wave number ranges (cf. also Giesbrecht, P., Naumann, D., Labi schi nski, H., in: Habermehl: Methods and Automation in Microbiology and Immunology, pp. 198-206, Berlin, Springer Verlag (1985); Naumann, D., Fijala, V., Labi schi nski, H ., Giesbrecht, P., J. Mol. Struct. 174, 165-170 (1988)). Despite powerful infrared spectroscopes and infrared microscopes, the sensitivity of these methods is not sufficient to be able to examine samples for the presence of microorganisms.

The object of the invention is to develop a method for the rapid detection of a very small number of microorganisms of the type mentioned at the beginning, and to make it possible to quickly and reliably detect a wide variety of microorganisms, even if the number of microorganisms in the sample to be examined is very small. According to the invention, this object is achieved by the measures specified in patent claim 1. The invention achieves the advantages of the various process groups used to date, namely detection of low microbial counts of microorganisms, possibility of connection with identification processes, low expenditure of time, broad applicability to all types of organism, if possible, using a uniform, easy-to-automate process, combined in one process. At the same time, the method according to the invention can also be effectively used in the practice of microbiological laboratories in routine operation. The spatially separated growth of microcolonies, starting from individual individuals of the microorganisms directly from the sample to be examined on the culture medium and the examination of a surface sample obtained from the culture medium after true-to-location transmission, does this according to the invention Processes are particularly suitable and valuable for examining samples which should be free of microorganisms or have only a very small number of bacteria.

The growth time according to the invention of at least 6 generation times of the organisms to be detected is based on the consideration that a localized concentration of the respective microorganism on a solid support for the sample, in particular on an agar plate, ^" enriches the microorganism up to one such degrees that the colonies formed are sufficiently large for detection with a microscope, in particular an IR microscope, and an ΪR spectroscope due to the low concentration of the microorganisms in the sample to be examined, possibly after a possible one Dilution, it is ensured that the individual microorganisms are applied as individuals in a large spatial separation to the surface of the solid support, so that the resulting colonies of the microorganisms do not adversely affect one another - Organisms in the microscopic range are carried out directly from the sample, without prior or subsequent isolation of the individual microorganisms being necessary. The size of the colonies of the microorganisms should be such that the respective colony has an area which corresponds to a number of at least about 50 individuals, for example bacteria. Starting from a single cell, 64 cells are formed in 6 generation times, the area of which is already sufficient for infrared spectroscopy. In the case of particularly large cells, smaller cells 1, such as 2, may also be sufficient. For security reasons, however, the colony is generally used with a larger surface area, which is achieved in particular by at least 7-12 generation times. 10 generation times correspond to approximately 1000 cells.

The length of time within which colonial growth is achieved depends on the growth rate of the individual microorganisms. A generation time of 20-40 minutes is normally sufficient for samples taken from the clinical area and a generation time of approximately one hour for water samples. In order to be sure, the period of time is kept in practice at about 4 to 10 hours. The time required and suitable can also be determined by preliminary tests. It is thereby achieved that the routine application of the method can be carried out with the shortest possible time period in order to achieve the desired result as quickly as possible. Mixing of constituents of different colonies is avoided by the accurate transfer of surface layers of the microcolonies obtained to the optical support. This also makes the method according to the invention suitable for enumeration and identification of the microorganisms. A high optical resolution and meaningfulness are achieved by using diaphragms of a suitable diameter. A particularly suitable device for carrying out the method according to the invention is the subject of claim 10. Advantageous further developments and features of the method and the device according to the invention are apparent from the subclaims in conjunction with the following description. The individual features can be implemented individually or in combination with one another. ^•

Of particular advantage is the preferred use of infrared microscopes, which can be reproducibly measured down to below the nanogram range by the further development of the Fourier transform infrared spectroscopy. The newly developed techniques for transferring samples from microcolonies or microorganisms without prior pure culture, in particular directly onto infrared-transmissive or reflecting carrier materials, which can be measured directly in the optical and infrared-spectroscopic mode of the IR microscope, make detection particularly fast and reliable enables. Surprisingly, detection is already possible with the preferred use of one or more, even very small, sections (for example only a few 100 wavenumbers) from the overall spectrum, so that carrier materials can also be used which only have IR in very small wavenumber ranges - are transparent or reflective. The use of a stamp impression method for transferring samples from a culture medium usually used in bacteriology, in particular an agar culture plate, where in particular a circular stamp made of IR-transparent material of suitable thickness, for example, proved to be particularly advantageous for rapid detection With the help of a guide, if necessary also directly with the hand *, the culture plate is briefly pressed (for example for less than a second) and then lifted off again, parts of which are on the culture plate (after a brief incubation, for example a urine sample) ) located microcolonies of the possibly also different microorganisms, which are usually not yet perceptible to the naked eye, can be transferred to the stamping plate so that the impression samples on the stamping plate are directly on the sample table of the stamping plate without any further preparation effort FT-IR microscopes can be measured. This procedure also has the advantage that, at the operator's request, the incubation of the original culture plates can be continued for later control purposes. The transfer to the stamp serving as an optical carrier can also take place by pressing the movable culture carrier against the stamp surface.

An additional advantage is the fact that the microscope, in particular the light-optical mode of a preferably used IR microscope (possibly supported by methods of image evaluation with a television camera attached, for example, with or without further image processing) not only to control the interesting ones Areas of the stamp, but also for the preliminary assessment of the sample (for example according to the morphology of the microcolonies, color, shape of the microorganisms (cocci, rods etc.) can be used if desired. The actual, reliable detection is then carried out by Recording the corresponding infrared spectra which, owing to the basic chemical composition (cell wall, membrane, protein, ribosomes, nucleic acids, etc.) common to all microorganisms, have a spectral characteristic which is in principle similar for all microorganisms in question, so that the methodology, as desired, without practically all microorganisms e restriction can be applied. It proves to be a particular advantage of the method that the IR spectra of microorganisms, regardless of their basic similarity as a result of the specific chemical composition of the cells of individual types of microorganisms, can, if desired, also differentiate without additional equipment identification of the measured microorganisms by comparing the spectra with those of a suitable reference file down to the subspecies level can be carried out using a method previously described (Giesbrecht, P., Naumann, D., Labi schi nski, H., in: Habermehl: Methods and Automation in Microbiology and Immunology, pp. 198-206, Berlin: Springe Verlag (1985); Naumann, D., Fijala, V., Labi schinski, H., Giesbrecht, P., J. Mol. Struct. V74 , 165-170 (1988)), to which reference is made here.

According to the invention, as already mentioned, it is possible to carry out an identification of microorganisms in addition to the detection. The device according to the invention can, however, advantageously be designed such that all three main tasks, e.g. Detection, identification and, if necessary, even sensitivity tests can be carried out, so that an old goal of a uniform technology with a basically uniform operation for any microorganisms, which cannot be achieved with the previous state of the art, can now be achieved. In this context, it proves to be very advantageous to fully automate the entire process sequence from sample preparation and sample measurement to output of the result, if desired even including subsequent processing of the result, in the sense of an identification or sensitivity test since the method can in principle be carried out uniformly for all microorganisms in question and the technique of spectral registration using the FT-IR microscopic technique delivers a digital measurement result from the outset and is therefore easy to process electronically.

When a preferred embodiment of the method according to the invention is carried out, the sample to be examined, such as urine, blood, liquor, water, aqueous extracts, is extracted Foodstuffs, ointments, pharmaceuticals, etc., diluted in the manner customary in microbiology onto a solid culture medium plate in the manner known from microbiology, such that the microorganisms which may be present in the sample grow into colonies in isolated cases. After a short cultivation time of about 4-10 hours (depending on the type of sample and the generation time of the microorganisms to be detected), during which M1 crocol onions not yet detectable with the eye from, for example, approx.

3 10 organisms have formed, the transfer takes place from the

Culture plate on the transparent or reflective support in the desired wavenumber range, for example by placing a sample support disk of, for example, about 2 mm thick and 3 cm in diameter made of, for example, ZnSe, polished steel, etc., and then lifting it again, so that the sample support a local impression of adhering microorganisms from the culture plate results. The sample carrier is then transferred to the sample table of the microscope, in particular an IR microscope, in a manually or automatically controlled manner. At this point in the process, several variants are possible, depending on the experimenter's wishes and the details of the microscope. In the simplest case, the sample plate can be inspected manually / visually in the optical mode of the microscope. From the number of optically recognizable microcolonies (taking into account possible dilution factors depending on the sample dilution in the manner customary in microbiology), an initial estimate of the number of bacteria present in the sample can be made from the colony morphology (size , Color, surface quality, etc.) may already be a rough estimate of possibly different organism groups. If no microcolonies can be identified at this point in time, the process can already be ended with the result "negative" at this point. The actual detection then takes place in the infrared spectroscopic mode of the microscope, in that of all or of a representative one The number of microcolonies on the sample carrier are recorded in succession with the aid of the aperture systems integrated in each IR microscope, and the infrared spectra of the corresponding microcolonies are recorded. Only those detected optically previously Mi krokoloni e-prints, ^* whose Spek¬ centers with the typical microorganisms chemical Zusam¬ mensetzung (especially proteins Liplde, nucleic acids, polysaccharides) are compatible, are finally accepted as detected. In this way it is prevented that imprint spots recognized in the optical mode, which originate, for example, from inorganic dirt (dust etc.) or also, for example, from transfers of media or agar components, contribute incorrectly to the result. The spectra obtained can, if desired, in the sense of a process variant, immediately afterwards or at a later time by comparing them with a spectra reference file in defo or the desired, even very small spectral range (s), according to the published process principles (see above) identification of the microorganisms can be used. In order, if desired, to make the process more independent of human intervention in the process, as described in principle above, a number of further process modifications are possible. For example, the intended transfer steps (ie a) application of the sample to the culture plate, b) transfer of the inoculated culture plate into an incubator, c) stamp impression for transferring the microcolonies from the culture carrier to the optical sample carrier, d) transfer if necessary of the sample carrier on the IR-M1 roskop-sample table and e) removal of the sample after the measurement has been carried out) easily mechanized and / or automated with the aid of conventional mechanical and / or electronic components. An additional, complete automatic process handling can be achieved by adding an image processing system consisting of a camera flanged onto the IR microscope and an electronically - 1 2 -

controlled X-Y cross table as a microscope table, an electronically controlled switchover unit between optical and IR microscopic mode of the microscope, a control or computer unit (e.g. personal computer with corresponding interfaces or other computer systems) and one or more corresponding evaluation programs. The computer and control unit, including evaluation programs, can expediently be integrated into the computer / control unit which is used from the outset to control the FT-IR device, including IR microscope.

A fully automatic process sequence then takes the following form: After the transfer of the sample carrier containing the microcolony impressions to the XY table of the IR microscope, an image of the carrier in the optical mode of the microscope can be recorded and digitized by the camera ( in at least 256 x 256 pixels, for example 64 gray levels) are passed on to the computer / control unit. The XY positions of possible microcolony impressions can be determined there by evaluating the digital gray scale distribution of the image, for example using a peak search algorithm. Subsequently, the control unit can retract a suitable aperture (diameter, for example, 30-80 μm), position it on the spatial coordinates of the first suspected microcolon imprint, and register the infrared spectrum at this point. This process can be repeated until all previously determined XY positions have been processed. The control unit can then initiate the call of a further subroutine which checks all the spectra registered in this way for their compatibility with microorganism spectra. This can be achieved, for example, by means of a simple cross-correlation, if necessary with prior filtering of the spectrums with reference spectra of microorganisms, since a threshold value for each individual but also combined spectra range with a given filter method of the correlation coefficient between microorganism spectra exists, which, if undershot, identifies the suspected microcolony as inorganic or organic contamination of the sample carrier. A reference number of 5-20 typical microorganism spectra (e.g. Staphyl kokkeπ, streptococci, clostridia, pseudomonas, Entero bacteri aceen, Salmonella, Legionella, bacilli) is sufficient for safe discrimination between microorganisms and contamination. Of course, peak tables or, for example, an evaluation using known methods of multivariate statistics can also be used for special applications. The number of microorganisms present in the sample can then be calculated and output by the control / arithmetic unit based on the number of positions confirmed as microcolony impressions, possibly taking into account corresponding dilution factors, for example as the number of bacteria / ml sample. With the help of the control and arithmetic unit k, if desired, with appropriate programming, subsequent identification of the detected microorganisms can also be made by comparison with a reference file v, which consists of reference spectra measured under the same conditions. The required program parts are based on algorithms well known to the person skilled in the art and can be created by yourself or from a large number of generally available program systems (the IMAGIC program, for example, has been successfully used in this context, see M. van Heel, W. Keegstra, Ultramicroscopy 7, 113, 1981).

Overall, the new method for the detection of microorganisms in the manner described allows rapid qualitative and quantitative detection of microorganisms in principle of all types with the further advantage of additional identification with one and the same basic apparatus in a time of approximately 4-10 hours or less after dispensing samples without prior culture, this time duration is almost completely determined by the necessary incubation time - until M1 microcolons are obtained, since the further process steps only take minutes to less than an hour if the process is carried out appropriately.

A Grobübersi CHT over the entire process flow of the preferred embodiments showing Fi _g. 1 in a schematic representation.

example 1

This example 1, in conjunction with FIG. 2, is intended to demonstrate the extraordinary sensitivity of the new method without describing the entire procedure. 2 shows three spectra obtained in the following manner with an infrared microscope A 560, from Bruker, Karlsruhe, coupled to an IFS 48 FT-IR spectrometer from the same company:

3 100 μl of a culture of Klebsiella pneu iniae, ATCC 13882, containing 10 germs / ml, in peptone water was plated onto a peptone agar plate and incubated for 8 hours at 37 ° C. (approx. 24 generation times). The bacteria were imprinted by carefully placing and removing a polished BaF® sample carrier of 40 mm in diameter and 3 mm in thickness. The bacterial film that is transferred, usually (in particular with shorter incubation times) and consisting of 1-3 bacterial layers, dries within a few seconds at room conditions without the use of vacuum or drying agents. The inspection of the sample carrier in the optical mode of the IR microscope (objective 15x, Casse-graniaπ, eyepiece 20x) revealed an extensive, thin, l-31-layer bacterial film over practically the entire sample carrier surface. Through the selection of suitable circular apertures, it was achieved that in each case approx. 10 (spectra 1 in FIG. 2, aperture 80 μm diameter), approx. 10 (spectra 2, aperture 40 μm diameter) or approx. 10 (spectra 3, aperture 20 μ diameter) microorganisms contribute to the spectrum. All spectra were measured in transmission, 512 scans were accumulated with a spectral resolution of 8 cm. Inspection of FIG. 2 clearly shows that already with approx. 10 microorganisms (corresponds to less than 0.1 ng substance) spectral signals to be detected clearly as microorganism spectra, shown here in the range of approx. 4000-90 wave numbers, are too are preserved. Due to the relatively high noise level 1 s (cf. spectra train 3 n FIG. 2), however, working with at least about 10

Microorganisms are to be preferred if the most detailed possible, eg species identification, is to follow the detection with the same data set. It can be seen from this typical measurement example that the incubation time of the sample on the culture plate should be at least approximately 6 to 12 ^' generation times in order to obtain a microcolony of approximately 200 from each microorganism isolated on the culture plate by correspondingly diluted application - To create 4000 organisms. Thus, for example, for enterobacteriaces (typical generation time on ^" usual media approx. 20 minutes) there is a lower limit for the incubation cell space of less than 4 hours, for staphylococci (generation time on usual media eg 30 minutes) of approx 5 hours etc.

Example 2

This example shows a typical process sequence when carrying out a manual experiment using the example of an aqueous sample which has been artificially mixed with 10 cells / ml of Staphylococcus aureus strain Pelzer. 100 μl of this sample were applied to a Peptoπ agar plate (100 mm in diameter) and distributed using a Drigal sk1 spatula. After an 8-hour incubation period at 37 ° C in an incubator the microcolonies (not yet visible to the naked eye) were transferred to a 40 mm BaF ₂ plate in the manner described in Example 1 (stamp impression).

Fig. 3 shows an enlarged image (250x) of a section of the sample holder. Of the in <Fig. 3 shown three Mi krokol onie-PRINT spectra obtained in the range 1800 to 800 cm are in ^'Fig. 4 dar¬ provided. The visual comparison of the three spectra already shows the excellent reproducibility of the method by - recording different microcolonies of the same microorganism. For quantitative confirmation, the so-called difference index D (D = (1- oC) ^• 1000, σc = Pearson's correlation coefficient) was determined by cross correlation. It was obtained in the measurements shown here, as well as in the case of other species, for D « ⁵ » 8, taking measurements of independently prepared samples, which were prepared at different times on agar plates of different batches of the same nutrient medium. Thus, the D-value of approx. 8 (corresponding to a correlation coefficient of 0.9921) determines the surprisingly good level of reproduction of the method and, in the event of subsequent desired differentiation or identification of the detected microorganisms, provides the pressure value for differentiating between different microorganisms. In the example described, 17 microcolony prints were detected on the carrier plate via their infrared spectrum. From this value, taking into account the amount of sample used (100 μl) and the area ratio of the agar plate to the sample carrier area (6.26: 1), an excellent depletion of tea KKeeiimmzzaahll vvoonn 11..0066xx1100 in excellent agreement with the value used. In game 3

This example illustrates the procedure of an identification following the detection. found

Mi kroorgani s en:

3 100 μl of a water sample with 10 germs per ml of

Staphylococcus aureus, Staphylococcus xylόsus and Klebsiell pneu oniae were applied to a peptone-agar plate as in Example 2, after an incubation time of 8 hours and with the same stamp technique applied to the same sample carrier as described in Example 2. 3 shows a 250x enlarged section of the sample carrier. Because of the location-specific and therefore area-precise transmission of the microcolonies, the observation in the optical mode of the microscope already gives indications for the involvement of several organisms, even for an experimentally trained bacteriologist, since the different microcolony sizes, as shown in Fig 4 shows three different microorganisms. At higher magnifications, the single bacterial morphology (eg cocci or rods) can also be used. For the experimenter, however, this is only one possible additional aid, because in the course of the method the detection is carried out by registering the corresponding infrared spectra. . The be¬ for it in Fig 5 _m 1, 2 and 3 recorded Mi krokol onien Footprints obtained spectra in the infrared mode of the FT-IR-Mi electron microscope shows' Fig. * - 6. All spectra show the characteristic over Pr benverl on the presence of microorganisms. The comparison in Fig. 6 with spectra trains shown denejι _etc. Re¬ a conference file 100 spectra of bacteria from 4 $ .ve lack e- those species found on the basis of the lowest TD-values calculated from the first derivatives of the spectra in the Wavenumber range from 1500-900 cm ^" , a species-specific identification for all microcolony prints. Examples and comments on FIG. 1

(a) e.g. Urine, cerebrospinal fluid, blood, food, water, etc., if necessary after appropriate preparation and dilution

(b) e.g. Agar plate with blood additive, peptone or the like, possibly selective or elective medium

(c) e.g. 20-40 degrees Celsius, pH 4-9, for e.g. 4-10 hours or about 8-20 generation times of the microorganisms

⁽ d ⁾ e.g. stamp imprint on ZnSe, BaF ₂ _ _Scll eiben, ^' polymer film, aluminum plate, polished steel plate etc.

(e) e.g. Manual, visual or automatic scanning of the sample carrier, registration and, if necessary, electronic storage of impression position coordinates, possibly using image processing techniques, which provide information about the number, position and size of the colony impressions

(f) e.g. Serial, manual or automatically controlled approach to the impression coordinates determined in the previous step, swiveling in a suitable aperture in the range of approx.

10 - 200 µm and recording of the spectra in the wavenumber range of e.g. 5000 - 500 in the middle infrared

(g) e.g. optical spectra assessment by an experimenter, calculation of correlation coefficients for reference spectra, multivariate analysis of the spectra etc.

(h) identification if necessary by comparison with reference data

⁽ i ⁾ if necessary taking into account sample dilution etc., additional results such as identification, colonial shape etc.

Claims

Method for the rapid detection of microorganisms, such as bacteria and fungi, in samples, characterized in that a sample to be examined for microorganisms is applied in a dilution to an essentially solid culture carrier used in microbiology is to allow the nien ^* growth vorhande¬ ner microorganisms in physically separate Kolo¬, b) a short-term growth of at least 6 Genera¬ tion times of the organisms to be detected to separate locally Mikrokol onien under the usual conditions in microbiology is caused

Erεat biatt c) surface areas of the culture carrier having microcolonies are transferred to an optical carrier that is either at least partially transparent or reflective in the desired spectral range, d) individual colonies of the transferred microorganisms are located in a microscope and e) the transferred microorganism colonies IR spectra can be recorded and evaluated individually individually using an infrared microscope in transmission or reflection mode using diaphragms in the diameter range of approximately 10-200 μm.

2. The method according to claim 1, characterized in that an exclusion of components which may be transferred to the carrier and which do not represent microorganisms, in particular organic and / or inorganic contaminants, nutrient components and / or dust, by a Comparison of the spectra obtained with spectra of reference microorganisms is carried out.

3. The method according to any one of the preceding claims, characterized in that the localization of the microorganism colonies in an optical or IR spectroscopy see operating mode of the infrared microscope is carried out.

4. The method according to any one of the preceding claims, characterized in that the true-to-life transmission of the microorganisms from the culture carrier is carried out in a direct manner on a stamp whose stamp surface consists of a material which is transparent or reflective in the desired wave range.

5. The method according to any one of the preceding claims, characterized in that the microorganism sample is transferred from the culture medium to a stamp by simple printing and the stamp is then used as an optical sample carrier and in particular as a cuvette part in the IR device or microscope Measurement of the spectra is transferred.

6. The method according to any one of the preceding claims, characterized in that the infrared spectra in one or more, possibly very small wave number intervals of e.g. less than 300 cm, or only individual, selective absorption bands characteristic of certain microorganisms or band contours in the NIR, MIR and / or FIR range of the infrared spectrum are measured.

7. The method according to any one of the preceding claims, characterized in that the evaluation of the spectra for bacterial detection by means of mathematical techniques of data reduction by methods of ulivariate statistics, calculation of correlation coefficients and similar comparative measures to reference spectra etc. with or without the aid mathematical techniques such as Spectra smoothing, spectral filtering or the like is carried out.

8. The method according to any one of the preceding claims, characterized in that, in addition to the pure detection of microorganisms, the same is additionally carried out by comparison with spectra reference files. ^"*

9. The method according to any one of the preceding claims, characterized in that for the enumeration of the microorganisms, the number of per unit area found microorganism colonies and back calculated on the sample, the counting of different colonies preferably being carried out separately.

10. Device for carrying out the method according to one of the preceding claims, characterized by coupling and / or integration a) an inoculation unit for placing the samples on the culture carrier, b) an incubator unit for incubating the cultures, c) a transfer unit for transferring ge Microcolonies on an optical carrier, d) a unit for localizing microorganism colonies by means of a microscope, e) an IR device and f) a unit for the controlled control of the individual functions of the apparatus.

11. The device according to claim 10, characterized in that the microscope is an IR microscope, in particular an FT-IR microscope.

12. The apparatus of claim 10 or 11, further characterized by a transfer unit for transferring the optical carrier to the stage of a microscope, in particular IR microscope, a mechanical unit for scanning the transmitted sample using the optical mode an IR microscope and / or a detection unit for recording micro colony on the optical carrier in the optical and infrared spectroscopy see mode of an IR microscope.

13. Device according to one of claims 10 to 12, characterized in that the control unit of the apparatus is set up in parallel or alternatively for detection also for controlling an identification.

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